Sucrose phosphorylase
Encyclopedia
Sucrose phosphorylase is an important enzyme in the metabolism of sucrose
and regulation of other metabolic intermediates. Sucrose phosphorylase is in the class of hexosyltransferases
. More specifically it has been placed in the retaining glycoside hydrolases family although it catalyzes a transglycosidation rather than hydrolysis. Sucrose phosphorylase catalyzes the conversion of sucrose to D-fructose and α-D-glucose-1-phosphate
(Reid and Abratt 2005). It has been shown in multiple experiments that the enzyme catalyzes this conversion by a double displacement mechanism.
is released by the enzyme-substrate complex. A covalent glucose-enzyme complex results, with beta-linkage between an oxygen atom in the carboxyl group of an aspartyl residue and C-1 of glucose. The covalent complex was experimentally isolated by chemical modification of the protein using NaIO4 after addition of the substrate
(Voet and Abeles, 1970; Mirza et al. 2006), supporting the hypothesis that reaction catalyzed by sucrose phosphorylase proceeds through the ping-pong mechanism. In the final enzymatic step, the glycosidic bond
is cleaved through reaction with a phosphate
group, yielding α-D-glucose-1-phosphate.
In a separate reaction, α-D-glucose-1-phosphate is converted to glucose-6-phosphate
by the action of phosphoglucomutase
(Tedokon et al. 1992). Glucose-6-phosphate is an extremely important intermediate for several pathways in the human body, including glycolysis
, gluconeogenesis
, and the pentose phosphate pathway
(Nelson and Cox 2005). The function of sucrose phosphorylase is especially significant due to the role α-D-glucose-1-phosphate in energy metabolism.
, was found to be 55 KDa, consisting of 488 amino acids (Koga et al. 1991). The active has been shown to contain two binding sites, one designated a water site where hydroxylic molecules such as 1,2-cyclohexanediol and ethylene glycol
may bind, and another designated as the acceptor site where the sugar molecule binds. Though the function of the water site has not been completely elucidated, the enzyme’s stability in aqueous solutions indicates that the water site may be involved in hydrolysis of the glycosidic bond.
The acceptor site is surrounded by three active residues that have been found to be essential in enzymatic activity. Using specific mutagenic assays, Asp-192 was found to be the catalytic nucleophile
of the enzyme, “attacking C-1 of the glucosyl moiety of sucrose” (Schwarz and Nidetzky 2006). In fact, in vitro
manipulation has shown that D-xylose
, L-sorbose
, and L-arabinose
can replace fructose as the glucosyl acceptor (Mieyal, Simon, and Abeles 1972). The only requirement of the acceptor molecule is that the hydroxyl group on the C-3 be cis-disposed to the oxygen atom of the glycosidic bond. Glu-232 acts as the Bronsted acid-base catalyst, donating a proton to the displaced hydroxyl group on C-1 of the glucoside (Schwarz, Brecker, and Nidetzky 2007).
The most significant residue in the enzymatic activity, however, is Asp-295 (Mueller and Nidetzky 2007). Upon cleavage of the fructofuranosyl moiety from sucrose, the resultant glucose forms a covalent intermediate with the enzyme. The carboxylate side chain of Asp-295 hydrogen bonds with the hydroxyl groups at C-2 and C-3 of the glucosyl residue (Mueller and Nidetzky 2007). This interaction is maximized during the transition state
of this covalent complex, lending support to the ping-pong mechanism. Finally, phosphorylation of the glucosyl residue at C-1 forms a transient positive charge on the glucosyl carbon, promoting breakage of the ester bond between Asp-192 and the sugar residue (Schwarz and Nidetzky 2006). Cleavage yields the product, α-D-glucose-1-phosphate.
al, affecting the amount of enzyme present at any given time.
Global regulation of DNA molecules containing the gene for sucrose phosphorylase is performed by catabolite repression
. First discovered in Gram-negative
bacteria, both Cyclic AMP (cAMP) and cAMP Receptor Protein
(CRP) function in sucrose phosphorylase regulation (Reid and Abratt 2005). The cAMP-CRP complex formed when both molecules combine acts as a positive regulator for transcription of the sucrose phosphorylase gene. The complex binds to the promoter region to activate transcription, enhancing the creation of sucrose phosphorylase (Nelson and Cox 2005).
Genetic regulation of sucrose phosphorylase is also performed by metabolites. Through experimentation it is known that genes encoding for the sucrose phosphorylase enzyme can be induced by sucrose and raffinose
(Trindade, Abratt, and Reid 2003). Glucose
, on the other hand, represses the transcription of the sucrose phosphorylase gene (Trindade, Abratt, and Reid 2003). These metabolites undoubtedly function in this way because of their implications in cellular metabolism.
There has been little research on methods of the allosteric regulation
of sucrose phosphorylase, so at this point the function of allosteric molecules can only be hypothesized. Due to the nature of its function in metabolic pathways, it is likely that sucrose phosphorylase is additionally regulated by other common metabolites. For example, the presence of ATP
would probably inhibit sucrose phosphorylase since ATP is a product of the catabolic pathway. Conversely, ADP
would likely stimulate sucrose phosphorylase to increase levels of ATP. Further research on the subject would be required to support or refute these ideas.
to glucose-6-phosphate (Tedokon et al. 1992), which is an important intermediate used in glycolysis. In addition, fructose can be reversibly converted into fructose 6-phosphate
(Reid and Abratt 2005), also found in the glycolytic pathway. In fact, fructose-6-phosphate and glucose-6-phosphate can be interconverted in the glycolytic pathway by phosphohexose isomerase (Nelson and Cox 2005). The final product of glycolysis, pyruvate, has multiple implications in metabolism. During anaerobic conditions, pyruvate con be converted into either lactate
or ethanol
, depending on the organism, providing a quick source of energy. In aerobic conditions,
pyruvate can be converted into Acetyl-CoA
, which has many possible fates including catabolism
in the Citric Acid Cycle
for energy use and anabolism
in the formation of fatty acids for energy storage. Through these reactions, sucrose phosphorylase becomes important in the regulation of metabolic functions.
The regulation of sucrose phosphorylase can also be used to explain its function in terms of energy consumption and preservation. The cAMP-CRP complex that enhances transcription of the sucrose phosphorylase gene (Reid and Abratt 2003) is only present when glucose levels are low. The purpose of sucrose phosphorylase, therefore, can be linked to the need for higher glucose levels, created through its reaction. The fact that glucose acts as a feedback inhibitor
to prevent the formation of sucrose phosphorylase (Reid and Abratt 2005) further supports its catalytic role in the creation of glucose for energy use or storage.
The glucose-6-phosphate molecule created from the original α-D-glucose-1-phosphate product is also involved in the pentose phosphate pathway
. Through a series of reactions, glucose-6-phosphate can be converted into ribose-5-phosphate, which is used for a variety of molecules such as nucleotides, coenzymes, DNA
, and RNA
(Nelson and Cox 2005). These connections reveal that sucrose phosphorylase is also important for the regulation of other cellular molecules.
Sucrose
Sucrose is the organic compound commonly known as table sugar and sometimes called saccharose. A white, odorless, crystalline powder with a sweet taste, it is best known for its role in human nutrition. The molecule is a disaccharide composed of glucose and fructose with the molecular formula...
and regulation of other metabolic intermediates. Sucrose phosphorylase is in the class of hexosyltransferases
Hexosyltransferases
Hexosyltransferases are a type of glycosyltransferase that catalyze the transfer of a hexose.Examples include:* glucosyltransferases - transfer glucose* galactosyltransferases - transfer galactose* fucosyltransferases - transfer fucose...
. More specifically it has been placed in the retaining glycoside hydrolases family although it catalyzes a transglycosidation rather than hydrolysis. Sucrose phosphorylase catalyzes the conversion of sucrose to D-fructose and α-D-glucose-1-phosphate
Glucose-1-phosphate
Glucose 1-phosphate is a glucose molecule with a phosphate group on the 1'-carbon.-Catabolic:In glycogenolysis, it is the direct product of the reaction in which glycogen phosphorylase cleaves off a molecule of glucose from a greater glycogen structure.To be utilized in cellular catabolism it must...
(Reid and Abratt 2005). It has been shown in multiple experiments that the enzyme catalyzes this conversion by a double displacement mechanism.
Reaction
The method by which sucrose phosphorylase converts sucrose to D-fructose and alpha-D-glucose-1-phosphate has been studied in great detail. In the reaction, sucrose binds to the enzyme, at which point fructoseFructose
Fructose, or fruit sugar, is a simple monosaccharide found in many plants. It is one of the three dietary monosaccharides, along with glucose and galactose, that are absorbed directly into the bloodstream during digestion. Fructose was discovered by French chemist Augustin-Pierre Dubrunfaut in 1847...
is released by the enzyme-substrate complex. A covalent glucose-enzyme complex results, with beta-linkage between an oxygen atom in the carboxyl group of an aspartyl residue and C-1 of glucose. The covalent complex was experimentally isolated by chemical modification of the protein using NaIO4 after addition of the substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
(Voet and Abeles, 1970; Mirza et al. 2006), supporting the hypothesis that reaction catalyzed by sucrose phosphorylase proceeds through the ping-pong mechanism. In the final enzymatic step, the glycosidic bond
Glycosidic bond
In chemistry, a glycosidic bond is a type of covalent bond that joins a carbohydrate molecule to another group, which may or may not be another carbohydrate....
is cleaved through reaction with a phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
group, yielding α-D-glucose-1-phosphate.
In a separate reaction, α-D-glucose-1-phosphate is converted to glucose-6-phosphate
Glucose-6-phosphate
Glucose 6-phosphate is glucose sugar phosphorylated on carbon 6. This compound is very common in cells as the vast majority of glucose entering a cell will become phosphorylated in this way....
by the action of phosphoglucomutase
Phosphoglucomutase
Phosphoglucomutase is an enzyme that transfers a phosphate group on an α-D-glucose monomer from the 1' to the 6' position in the forward direction or the 6' to the 1' position in the reverse direction....
(Tedokon et al. 1992). Glucose-6-phosphate is an extremely important intermediate for several pathways in the human body, including glycolysis
Glycolysis
Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...
, gluconeogenesis
Gluconeogenesis
Gluconeogenesis is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as lactate, glycerol, and glucogenic amino acids....
, and the pentose phosphate pathway
Pentose phosphate pathway
The pentose phosphate pathway is a process that generates NADPH and pentoses . There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH is generated, and the second is the non-oxidative synthesis of 5-carbon sugars...
(Nelson and Cox 2005). The function of sucrose phosphorylase is especially significant due to the role α-D-glucose-1-phosphate in energy metabolism.
Structural Implications
The structure of sucrose phosphorylase has been identified in numerous experiments. The enzyme consists of four major domains, namely A, B, B’, and C. Domains A, B’ and C exist as dimers around the active site (Sprogoe et al. 2004). The size of the enzyme, as determined by sedimentation centrifugationCentrifugation
Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...
, was found to be 55 KDa, consisting of 488 amino acids (Koga et al. 1991). The active has been shown to contain two binding sites, one designated a water site where hydroxylic molecules such as 1,2-cyclohexanediol and ethylene glycol
Ethylene glycol
Ethylene glycol is an organic compound widely used as an automotive antifreeze and a precursor to polymers. In its pure form, it is an odorless, colorless, syrupy, sweet-tasting liquid...
may bind, and another designated as the acceptor site where the sugar molecule binds. Though the function of the water site has not been completely elucidated, the enzyme’s stability in aqueous solutions indicates that the water site may be involved in hydrolysis of the glycosidic bond.
The acceptor site is surrounded by three active residues that have been found to be essential in enzymatic activity. Using specific mutagenic assays, Asp-192 was found to be the catalytic nucleophile
Nucleophile
A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction. All molecules or ions with a free pair of electrons can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.Nucleophilic describes the...
of the enzyme, “attacking C-1 of the glucosyl moiety of sucrose” (Schwarz and Nidetzky 2006). In fact, in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
manipulation has shown that D-xylose
Xylose
Xylose is a sugar first isolated from wood, and named for it. Xylose is classified as a monosaccharide of the aldopentose type, which means that it contains five carbon atoms and includes an aldehyde functional group. It is the precursor to hemicellulose, one of the main constituents of biomass...
, L-sorbose
Sorbose
Sorbose is a ketose belonging to the group of sugars known as monosaccharides. It has a sweetness that is equivalent to sucrose . The commercial production of vitamin C often begins with sorbose. L-Sorbose is the configuration of the naturally occurring sugar....
, and L-arabinose
Arabinose
Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde functional group.For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, or structurally analogous to D-glyceraldehyde.For sugars, the D/L...
can replace fructose as the glucosyl acceptor (Mieyal, Simon, and Abeles 1972). The only requirement of the acceptor molecule is that the hydroxyl group on the C-3 be cis-disposed to the oxygen atom of the glycosidic bond. Glu-232 acts as the Bronsted acid-base catalyst, donating a proton to the displaced hydroxyl group on C-1 of the glucoside (Schwarz, Brecker, and Nidetzky 2007).
The most significant residue in the enzymatic activity, however, is Asp-295 (Mueller and Nidetzky 2007). Upon cleavage of the fructofuranosyl moiety from sucrose, the resultant glucose forms a covalent intermediate with the enzyme. The carboxylate side chain of Asp-295 hydrogen bonds with the hydroxyl groups at C-2 and C-3 of the glucosyl residue (Mueller and Nidetzky 2007). This interaction is maximized during the transition state
Transition state
The transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...
of this covalent complex, lending support to the ping-pong mechanism. Finally, phosphorylation of the glucosyl residue at C-1 forms a transient positive charge on the glucosyl carbon, promoting breakage of the ester bond between Asp-192 and the sugar residue (Schwarz and Nidetzky 2006). Cleavage yields the product, α-D-glucose-1-phosphate.
Regulation
Since the discovery and characterization of sucrose phosphorylase, few documented experiments discuss mechanisms of regulation for the enzyme. The known methods of regulation are transcriptionTranscription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
al, affecting the amount of enzyme present at any given time.
Global regulation of DNA molecules containing the gene for sucrose phosphorylase is performed by catabolite repression
Catabolite repression
Carbon catabolite repression, or simply catabolite repression, is an important part of global control system of various bacteria and other micro-organisms. Catabolite repression allows bacteria to adapt quickly to a preferred carbon and energy source first...
. First discovered in Gram-negative
Gram-negative
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...
bacteria, both Cyclic AMP (cAMP) and cAMP Receptor Protein
CRP
-Biochemistry and medicine:* C-reactive protein , an acute phase protein produced by the liver* cAMP receptor protein is a regulatory protein in bacteria* Collagen-related peptides...
(CRP) function in sucrose phosphorylase regulation (Reid and Abratt 2005). The cAMP-CRP complex formed when both molecules combine acts as a positive regulator for transcription of the sucrose phosphorylase gene. The complex binds to the promoter region to activate transcription, enhancing the creation of sucrose phosphorylase (Nelson and Cox 2005).
Genetic regulation of sucrose phosphorylase is also performed by metabolites. Through experimentation it is known that genes encoding for the sucrose phosphorylase enzyme can be induced by sucrose and raffinose
Raffinose
Raffinose is a trisaccharide composed of galactose, fructose, and glucose. It can be found in beans, cabbage, brussels sprouts, broccoli, asparagus, other vegetables, and whole grains. Raffinose can be hydrolyzed to D-galactose and sucrose by the enzyme α-galactosidase , an enzyme not found in the...
(Trindade, Abratt, and Reid 2003). Glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
, on the other hand, represses the transcription of the sucrose phosphorylase gene (Trindade, Abratt, and Reid 2003). These metabolites undoubtedly function in this way because of their implications in cellular metabolism.
There has been little research on methods of the allosteric regulation
Allosteric regulation
In biochemistry, allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site . Effectors that enhance the protein's activity are referred to as allosteric activators, whereas those that decrease the protein's activity are...
of sucrose phosphorylase, so at this point the function of allosteric molecules can only be hypothesized. Due to the nature of its function in metabolic pathways, it is likely that sucrose phosphorylase is additionally regulated by other common metabolites. For example, the presence of ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
would probably inhibit sucrose phosphorylase since ATP is a product of the catabolic pathway. Conversely, ADP
Adenosine diphosphate
Adenosine diphosphate, abbreviated ADP, is a nucleoside diphosphate. It is an ester of pyrophosphoric acid with the nucleoside adenosine. ADP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase adenine....
would likely stimulate sucrose phosphorylase to increase levels of ATP. Further research on the subject would be required to support or refute these ideas.
Metabolic Function
As mentioned above, sucrose phosphorylase is a very important enzyme in metabolism. The reaction catalyzed by sucrose phosphorylase produces the valuable byproducts α-D-glucose-1-phosphate and fructose. α-D-glucose-1-phosphate can be reversibly converted by phosphoglucomutasePhosphoglucomutase
Phosphoglucomutase is an enzyme that transfers a phosphate group on an α-D-glucose monomer from the 1' to the 6' position in the forward direction or the 6' to the 1' position in the reverse direction....
to glucose-6-phosphate (Tedokon et al. 1992), which is an important intermediate used in glycolysis. In addition, fructose can be reversibly converted into fructose 6-phosphate
Fructose 6-phosphate
Fructose 6-phosphate is fructose sugar phosphorylated on carbon 6 . The β-D-form of this compound is very common in cells. The vast majority of glucose and fructose entering a cell will become converted to this at some point...
(Reid and Abratt 2005), also found in the glycolytic pathway. In fact, fructose-6-phosphate and glucose-6-phosphate can be interconverted in the glycolytic pathway by phosphohexose isomerase (Nelson and Cox 2005). The final product of glycolysis, pyruvate, has multiple implications in metabolism. During anaerobic conditions, pyruvate con be converted into either lactate
Lactic acid
Lactic acid, also known as milk acid, is a chemical compound that plays a role in various biochemical processes and was first isolated in 1780 by the Swedish chemist Carl Wilhelm Scheele. Lactic acid is a carboxylic acid with the chemical formula C3H6O3...
or ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
, depending on the organism, providing a quick source of energy. In aerobic conditions,
pyruvate can be converted into Acetyl-CoA
Acetyl-CoA
Acetyl coenzyme A or acetyl-CoA is an important molecule in metabolism, used in many biochemical reactions. Its main function is to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production. In chemical structure, acetyl-CoA is the thioester...
, which has many possible fates including catabolism
Catabolism
Catabolism is the set of metabolic pathways that break down molecules into smaller units and release energy. In catabolism, large molecules such as polysaccharides, lipids, nucleic acids and proteins are broken down into smaller units such as monosaccharides, fatty acids, nucleotides, and amino...
in the Citric Acid Cycle
Citric acid cycle
The citric acid cycle — also known as the tricarboxylic acid cycle , the Krebs cycle, or the Szent-Györgyi-Krebs cycle — is a series of chemical reactions which is used by all aerobic living organisms to generate energy through the oxidization of acetate derived from carbohydrates, fats and...
for energy use and anabolism
Anabolism
Anabolism is the set of metabolic pathways that construct molecules from smaller units. These reactions require energy. One way of categorizing metabolic processes, whether at the cellular, organ or organism level is as 'anabolic' or as 'catabolic', which is the opposite...
in the formation of fatty acids for energy storage. Through these reactions, sucrose phosphorylase becomes important in the regulation of metabolic functions.
The regulation of sucrose phosphorylase can also be used to explain its function in terms of energy consumption and preservation. The cAMP-CRP complex that enhances transcription of the sucrose phosphorylase gene (Reid and Abratt 2003) is only present when glucose levels are low. The purpose of sucrose phosphorylase, therefore, can be linked to the need for higher glucose levels, created through its reaction. The fact that glucose acts as a feedback inhibitor
Negative feedback
Negative feedback occurs when the output of a system acts to oppose changes to the input of the system, with the result that the changes are attenuated. If the overall feedback of the system is negative, then the system will tend to be stable.- Overview :...
to prevent the formation of sucrose phosphorylase (Reid and Abratt 2005) further supports its catalytic role in the creation of glucose for energy use or storage.
The glucose-6-phosphate molecule created from the original α-D-glucose-1-phosphate product is also involved in the pentose phosphate pathway
Pentose phosphate pathway
The pentose phosphate pathway is a process that generates NADPH and pentoses . There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH is generated, and the second is the non-oxidative synthesis of 5-carbon sugars...
. Through a series of reactions, glucose-6-phosphate can be converted into ribose-5-phosphate, which is used for a variety of molecules such as nucleotides, coenzymes, DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, and RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
(Nelson and Cox 2005). These connections reveal that sucrose phosphorylase is also important for the regulation of other cellular molecules.